White blood cells called T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. They are also involved in allergies/asthma due to the development of an unwanted or excessive type of immune response to substances (antigens) in our environment and in autoimmune diseases. Because T cells respond to foreign substances (antigens) in the form of peptide- major histocompatibility complex (MHC) molecule complexes on cell surfaces, we wish to know how such complexes interact with specific receptors to evoke the effector activities of mature T cells in the body, as well as regulate their growth, inactivation, or death. In particular, we want to understand in molecular detail the protein-protein interactions that turn recognition of antigen by T cells into signals guiding the normal survival and effector functions of these cells, how variations in these recognition and signaling events leads to desirable versus undesirable forms of immunity, and how we can manipulate these events to augment useful immune responses and inhibit damaging ones. Our studies currently focus on the biophysics of the binding of the T-cell receptor (TCR) and CD4 / CD8 coreceptors with peptide:MHC molecule ligands, on new biochemical regulatory pathways that help T-cells discriminate between self- and foreign peptide:MHC molecule complexes, and on the role of self-recognition in the sensitivity of T-cells to antigen on presenting cells of the types studied in LI545. During the past year we have extended our analysis of the molecular details of two novel regulatory pathways controlling early signaling by the T-cell receptor (SHP-1 phosphatase dependent negative control and MAPK mediated positive control). Using retroviral gene transduction we have documented the role of these pathways in the regulation of signaling and functional responses of normal CD4+ T-cells. We have also used confocal microscopy to demonstrate polarization of TCR and the pre-docking of critical signaling components in the TCR complex induced / maintained by self-MHC recognition. The laboratory has developed novel methods for the tracking of peptide/MHC molecule ligand binding to the TCR and interaction with CD8 coreceptors with a time resolution not previously possible, especially with living cells. Our studies of cytoskeletal adapter proteins (ERM molecules) have led us into experiments that have revealed the regulation of T-cell rigidity by TCR signals and the role of small GTPases (Rac, CDC42) in this regulatory process. We have extended our studies showing that recently activated T cells acquire overt reactivity to self antigens, which we believe helps expand clonal precursors during the period of antigen limitation early in an infectious process. Finally, we have modified or developed new computer programs that allow simulation in space and time of the behavior of lymphocytes in chemokine gradients and of receptors and signaling molecules on and below the cell membrane.